1. Field of the Invention
This invention relates to biocontrol agents for the prevention of aflatoxin contamination in corn, in particular, to certain non-toxigenic and non-aflatoxigenic Aspergillus flavus strains capable of inhibiting growth of fungi which produce aflatoxin, and which are further capable of suppressing production of aflatoxin by the toxigenic fungi. More specifically, the invention relates to the non-toxigenic Aspergillus flavus strain K49 (NRRL 30797) and the non-aflatoxigenic Aspergillus flavus strain CT3 (NRRL 30798) and their variants. The present invention relates to a biocontrol strategy whereby the A. flavus strains K49 and CT3 are applied to crops as a method for reducing aflatoxin contamination in corn, particularly in the Mississippi Delta.
2. Description of the Relevant Art
Aflatoxins are a class of mycotoxins produced by Aspergillus flavus Link and A. parasiticus Speare. There are four closely related aflatoxins: B1, B2, G1, and G2 (Council for Agricultural Science and Technology [CAST]. 2003. Task Force Report 139, Ames, Iowa; Diener et al. 1987. Ann. Rev. Phytopath. 25: 249-270). Toxigenic strains of A. flavus produce mainly B1 and B2, while A. parasiticus produces all four aflatoxins (CAST, 2003; Diener et al., supra). Aflatoxins are potent carcinogens and hepatotoxins. Aflatoxin contamination of corn (maize, Zea mays L.) is a significant problem worldwide (CAST, supra; Cullen et al. 1994. In: The Toxicology of Aflatoxins, Eaton et al. (Eds.), Academic Press, San Diego, Calif., pp. 3-26). United States federal guidelines for food and feed set a limit of 20 ng/g total aflatoxins, while the European Union guidelines are more strict, with a limit of 1 ng/g limit for B1 and a 4 ng/g limit for total aflatoxins (van Egmond, H. P. & Jonker, M. A. 2004. J. Toxicol.-Toxin Rev. 23: 273-293).
Cyclopiazonic acid (CPA) is another mycotoxin also produced by various Aspergillus species that may cause toxicological problems in animals (Bryden, W. L. 1991. In: Emerging Problems Resulting from Microbial Contamination, Mixe et al. (Eds.), National Institute of Hygienic Science, Tokyo, pp. 127-147). Cyclopiazonic acid has been found to occur in maize and other foods (Trucksess et al. 1987. J. Assoc. Official Analytical Chem. Int'l. 70: 123-126) and can further increase the risks of food and feed contaminated with A. flavus (Takahashi et al. 2004. J. Food Protect. 67: 90-95; Mphande et al. 2004. J. Food Protect. 67: 96-102; Sosa et al. 2002. J. Food Protect. 65: 988-992). Residues of CPA from feed can be transferred into milk and eggs (Dorner et al. 1983. Appl. Environ. Microbiol. 46: 698-703; CAST, supra). Strains of A. flavus vary greatly in aflatoxin production, with some producing copious amounts and others none (Abbas et al. 2004. J. Toxicol.-Toxin Rev. 23 (2,3): 153-450; Horn, B. W. 2003. J. Toxicol.-Toxin Rev. 22: 351-379). Many A. flavus strains produce both CPA and aflatoxins whereas other strains are non-toxigenic, i.e., the strains produce neither aflatoxins nor CPA. Other strains are non-aflatoxigenic, that is, they produce CPA, but do not produce aflatoxin (Horn and Dorner. 1999. Appl. Environ. Microbiol. 65: 1444-1449; Dorner, J. W. 2004. J. Toxicol.-Toxin Rev. 23: 425-450; Geiser et al. 2000. Fungal Genet. Biol. 31: 169-179).
Because aflatoxin contamination is a major economic and food safety concern, strategies have been developed to control aflatoxin in crops (Abbas, H. K. 2003. J. Toxicol.-Toxin Rev. 22 (2,3): 139-459; Abbas, H. K., 2004, supra; 2002. Aflatoxin/Fumonisin Elimination and Fungal Genomics Workshops, Phoenix, Ariz., Oct. 23-26, 2001, Robens, J. F. & Riley, R. T. (Eds.) Mycopathologia 155: 1-122; 2004. Aflatoxin/Fumonisin Elimination and Fungal Genomics Workshops, San Antonio, Tex., Oct. 23-26, 2002, Robens, J. F. & Brown, R. L. (Eds.) Mycopathologia 157: 393-505). Non-toxigenic strains of A. flavus have been suggested as biological control agents in hopes that they might compete with naturally-occurring toxigenic A. flavus. Early in vitro studies by Erhlich (Erhlich, K. 1987. Mycopathologia 97: 93-96) showed that co-inoculation of medium with a mixture of non-toxigenic mutants and the toxigenic wild-type significantly reduced aflatoxin contamination. The potential for biological control of aflatoxin has been demonstrated under field conditions in cotton (Gossypium hirsutum L.) and peanut (Arachis hypogaea L.). Cotty has shown that a non-toxigenic strain of A. flavus and a factor produced by a non-toxigenic strain of A. flavus can reduce aflatoxin contamination in cotton (Cotty, P. J. 1994a. Phytopath. 84: 1270-1277; Cotty, P. J. 1994b. U.S. Pat. No. 5,294,442, Mar. 15 1994; Cotty, P. J., U.S. Pat. No. 5,171,686, Dec. 15 1992). Others have utilized either a mixture of the non-toxigenic strains of A. parasiticus and A. flavus, oil formulations of these strains, or other Aspergillus strains: A. oryzae, A. sojae, and mixtures of A. oryzae and A. sojae, to reduce aflatoxin contamination in peanut (Cole et al. U.S. Pat. No. 5,292,661, Mar. 8 1994; Cole et al. U.S. Pat. No. 6,306,386, Oct. 23 2001; Dorner, J. W. and Cole, R. J. 2002. J. Stored Prod. Res. 38: 329-339: Dorner et al. U.S. Pat. No. 6,027,724, Feb. 22 2000).
Corn too is frequently infected by Aspergillus species which can result in significant aflatoxin accumulation, especially when heat and drought stress occur (Abbas et al. 2002. J. Agric. Food Chem. 50: 5246-5254; Payne, G. S. 1992. Crit. Rev. Plant Sci. 10: 423-440). When aflatoxin concentration exceeds regulatory levels, this contamination causes a severe economic impact on growers, the grain industry, and may be a significant health risk (Robens, J. & Cardwell, K. 2003. J. Toxicol.-Toxin Rev. 22: 139-152). Thus, suitable strategies to control aflatoxin contamination of corn have been sought to reduce the risks in corn production in certain geographical regions, such as the southern United States. In one study by Brown et al., individual ears of corn were wounded and inoculated directly and simultaneously with a spore solution containing toxigenic and atoxigenic A. flavus (1991. J. Food Protect. 54: 623-626). Brown et al. concluded that atoxigenic strains of A. flavus may have potential use as biological control agents to reduce aflatoxin contamination by toxigenic strains. Later studies by Dorner et al. (1999. J. Food Protect. 62: 650-656) evaluated the effect of crop rotation and inoculation of corn fields with rice infected with non-aflatoxigenic strains of A. flavus and A. parasiticus to determine the effect of application of the non-toxigenic strains on preharvest aflatoxin contamination of corn. Dorner et al. concluded that their study did not indicate that their biocontrol strategy offered a solution to the problem of aflatoxin contamination in corn (Page 655, last paragraph) and that inclusion of a non-aflatoxigenic strain of A. parasiticus in a biological control formulation for aflatoxin contamination may not be as important for airborne crops, such as corn, as for soilborne crops, such as peanuts (Page 650, Abstract).
In a survey of A. flavus from the Mississippi Delta region of the USA, we characterized over five hundred isolates of A. flavus from soil and various crops including corn, rice and peanuts (Abbas et al. 2004. Canad. J. Microbiol. 50: 193-199). Of these isolates, about 36% produced less than 20 ng/g total aflatoxins when grown on potato dextrose agar (PDA). As discussed above, field studies in cotton and peanuts have shown that non-toxigenic strains applied to soil are capable of competing and displacing naturally occurring toxigenic strains (Cotty, P. J., 1992, 1994a and 1994b, supra; Dorner, J. W. & Cole, R. J., 2002, supra; Dorner et al. U.S. 2000, supra; Cole et al., supra). While these various biocontrol methods and formulations for effective control of toxigenic fungi in cotton and peanuts are known in the art, there still remains a need for effective non-aflatoxigenic and non-toxigenic A. flavus strains to serve as biocontrol agents for controlling toxigenic A. flavus in corn. The present invention, described below, provides non-aflatoxigenic and non-toxigenic A. flavus strains and methods of using these strains to effectively reduce aflatoxin contamination in corn.